Circular RNA for translation in eukaryotic cells

Abstract

Circular RNA and methods and constructs for engineering circular RNA are disclosed. In some embodiments, the circular RNA includes the following elements arranged in the following sequence: a) a 3′ Group I self-splicing intron fragment, b) an internal ribosome entry site (IRES), c) a protein coding region or noncoding region, and d) a 5′ Group I self-splicing intron fragment.

What is claimed is: 
     
         1 . A circular RNA comprising the following elements arranged in the following sequence:
 a) an exon sequence of a 3′ Group I self-splicing intron-exon,   b) a 5′ internal homology region,   c) an internal ribosome entry site (IRES),   d) a protein coding region,   e) a 3′ internal homology region, and   f) an exon sequence of a 5′ Group I self-splicing intron-exon,   wherein the 5′ internal homology region, the 3′ internal homology region, or both are exogenous to the Group I self-splicing intron-exon.   
     
     
         2 . The circular RNA of  claim 1 , wherein the exon sequence of the 3′ Group I self-splicing intron-exon and the exon sequence of the 5′ Group I self-splicing intron-exon are from Cyanobacterium  Anabaena  or are from a Cyanobacterium  Anabaena  sp. pre-tRNA-Leu gene. 
     
     
         3 . The circular RNA of  claim 1 , further comprising:
 a) a 5′ spacer sequence comprising the 5′ internal homology region, and   b) a 3′ spacer sequence comprising the 3′ internal homology region.   
     
     
         4 . The circular RNA of  claim 3 , wherein:
 a) the 5′ spacer sequence is at least 7 nucleotides in length,   b) the 3′ spacer sequence is at least 7 nucleotides in length, or both a) and b).   
     
     
         5 . The circular RNA of  claim 3 , wherein:
 a) the 5′ spacer sequence is no more than 100 nucleotides in length,   b) the 3′ spacer sequence is no more than 100 nucleotides in length, or both a) and b).   
     
     
         6 . The circular RNA of  claim 3 , wherein:
 a) the 5′ spacer sequence is 10-60 nucleotides in length,   b) the 3′ spacer sequence is 10-60 nucleotides in length, or both a) and b).   
     
     
         7 . The circular RNA of  claim 3 , wherein:
 a) the 5′ spacer sequence comprises a polyA sequence, a polyC sequence, a polyA-C sequence, or a poly U sequence,   b) the 3′ spacer sequence comprises a polyA sequence, a polyC sequence, a polyA-C sequence, or a poly U sequence, or both a) and b).   
     
     
         8 . The circular RNA of  claim 1 , wherein the IRES is selected from an IRES sequence of Taura syndrome virus,  Triatoma  virus, Theiler's encephalomyelitis virus, simian Virus 40,  Solenopsis invicta  virus 1,  Rhopalosiphum padi  virus, Reticuloendotheliosis virus, human poliovirus 1,  Plautia stali  intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus (EMCV),  Drosophila  C Virus, Crucifer tobamo pvirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1,  Drosophila antennapedia , Human AQP4, Human ATIR, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3,  Drosophila  reaper, Canine Scamper,  Drosophila  Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP,  Drosophila  hairless,  S. cerevisiae  TFIID,  S. cerevisiae  YAP1, Human c-src, Human FGF-1, Simian picornavirus, Turnip crinkle virus, an aptamer to eIF4G, Coxsackievirus B3 (CVB3) or Coxsackievirus A (CVB1/2). 
     
     
         9 . The circular RNA of  claim 1 , wherein the IRES is an IRES sequence from Coxscakievirus B3 (CBV3), Encephalomyocarditis virus (EMCV), or Salivirus. 
     
     
         10 . The circular RNA of  claim 1 , wherein the circular RNA is at least 1,600 nucleotides in size. 
     
     
         11 . The circular RNA of  claim 1 , wherein the circular RNA is at least 1,700 nucleotides in size. 
     
     
         12 . The circular RNA of  claim 1 , wherein the protein coding region encodes a chimeric antigen receptor, a therapeutic protein, or a binding protein. 
     
     
         13 . The circular RNA of  claim 1 , wherein the circular RNA is no more than 5,000 nucleotides in size. 
     
     
         14 . A composition, comprising:
 a) an effective amount of a circular RNA, wherein the circular RNA comprises the following elements arranged in the following sequence:
 i) an exon sequence of a 3′ Group I self-splicing intron-exon, 
 ii) a 5′ internal homology region, 
 iii) an internal ribosome entry site (IRES), 
 iv) a protein coding region, 
 v) 3′ internal homology region, and 
 vi) an exon sequence of a 5′ Group I self-splicing intron-exon, wherein the 5′ internal homology region, the 3′ internal homology region, or both are exogenous to the Group I self-splicing intron-exon; and 
   b) a nanocarrier selected from the group consisting of a lipid, a polymer and a lipo-polymeric hybrid.   
     
     
         15 . The composition of  claim 14 , wherein the protein coding region encodes a chimeric antigen receptor, a therapeutic protein, or a binding protein. 
     
     
         16 . The composition of  claim 14 , wherein the circular RNA is at least 1,600 nucleotides in size. 
     
     
         17 . The composition of  claim 14 , wherein the circular RNA is at least 1,700 nucleotides in size. 
     
     
         18 . The composition of  claim 14 , wherein the circular RNA is at least 2,000 nucleotides in size. 
     
     
         19 . The composition of  claim 14 , wherein the circular RNA is no more than 5,000 nucleotides in size. 
     
     
         20 . A method of treating a disease, the method comprising administering an effective amount of a circular RNA to a subject in need thereof, wherein the circular RNA comprises the following elements arranged in the following sequence:
 a) an exon sequence of a 3′ Group I self-splicing intron-exon,   b) a 5′ internal homology region,   c) an internal ribosome entry site (IRES),   d) a protein coding region,   e) a 3′ internal homology region, and   f) an exon sequence of a 5′ Group I self-splicing intron-exon,   wherein the 5′ internal homology region, the 3′ internal homology region, or both are exogenous to the Group I self-splicing intron-exon.   
     
     
         21 . The method of  claim 20 , wherein the protein coding region encodes a chimeric antigen receptor, a therapeutic protein, or a binding protein.